The Development of Surface-Modified Liposomes as an Intranasal Delivery System for Group A Streptococcus Vaccines.

Intranasal vaccine administration can overcome the disadvantages of injectable vaccines and present greater efficiency for mass immunization. However, the development of intranasal vaccines is challenged by poor mucosal immunogenicity of antigens and the limited availability of mucosal adjuvants. Here, we examined a number of self-adjuvanting liposomal systems for intranasal delivery of lipopeptide vaccine against group A Streptococcus (GAS). Among them, two liposome formulations bearing lipidated cell-penetrating peptide KALA and a new lipidated chitosan derivative (oleoyl-quaternized chitosan, OTMC) stimulated high systemic antibody titers in outbred mice. The antibodies were fully functional and were able to kill GAS bacteria. Importantly, OTMC was far more effective at stimulating antibody production than the classical immune-stimulating trimethyl chitosan formulation. In a simple physical mixture, OTMC also enhanced the immune responses of the tested vaccine, without the need for a liposome delivery system. The adjuvanting capacity of OTMC was further confirmed by its ability to stimulate cytokine production by dendritic cells. Thus, we discovered a new immune stimulant with promising properties for mucosal vaccine development.

Tailoring a UCST-LCST-pH Multiresponsive Window through a Single Polymer Complex of Chitosan-Hyaluronic Acid.

Multistimuli-responsive polymers are important for controlled release. Owing to the fact that these polymers are derived from vinyl-based monomers, their decoration with other molecules is limited. Polysaccharides, especially chitosan (CS) and hyaluronic acid (HA), are pH-responsive biopolymers, whose chemical structures contain reactive functional groups for feasible chemical modifications to obtain add-on functions. The present work demonstrates the introduction of polymers with upper critical solution temperature (UCST) and lower critical solution temperature (LCST) performances onto CS and HA, respectively. By simply varying the mole ratio between the CS-containing UCST polymer and the HA-containing LCST polymer along with adjusting the pH, a polymer system with a UCST-LCST-pH multiresponsive window can be obtained. This multiresponsive window enables us to control the encapsulation and release with repeatability as evidenced from a model study on lysozyme. The present work, for the first time, shows a simple approach to obtain multiresponsive biodegradable polymers through the formation of a single polymer complex to tailor a specific multiresponsive window.

Nanocomplex of quaternized cyclodextrin grafted chitosan and hyaluronic acid for a skin delivery.

Water soluble quaternized cyclodexrin grafted chitosan (QCD-g-CS) was synthesized by combining both beneficial properties of ß-cyclodextrin (ß-CD) and the chitosan (CS) backbone. The chitosan backbone exhibits positive charges, while the ß-CD moieties are available to include hydrophobic guest molecules into the cavity. The present work demonstrates a formation of nanocomplexes by simple mixing of the cationic QCD-g-CS with three different molecular weights of anionic Hyaluronic acid (low, medium and high HA; LHA, MHA and HHA, respectively). The HA is well-known on providing hydration to the skin and normalize keratinization. However, its strong hydrophilicity limits skin absorption. The polyelectrolyte nanocomplexes between QCD-g-CS and HA formed through the electrostatic interactions were confirmed by FTIR. Particle size of HA nanocomplexes were greater than that of free QCD-g-CS and increased with an increase in HA content. The complex of LHA and MHA improve the water retention capacity as well as ability to control the release of HA to be slower than the original HA. The release of both LHA and MHA from their complexes were both limited diffusion kinetics. Pronounced effect of small particle sizes of LHA complexes was found to benefit skin penetration. Clinical study indicated that LHA complexes improved skin texture and elasticity due to an increase in skin hydration. It is suggested that the QCD-g-CS in combination with anionic hydrophilic HA can be used as a promising polysaccharide-based skin delivery system.

Brush-Structured Chitosan/PolyHEMA with Thymine and Its Synergistic Effect on the Specific Interaction with ssDNA and Cellular Uptake.

Cationic polymers are known to attach on an anionic cell surface and favor gene transportation/transfection into the cells. However, when the positive charges accumulate, they tend to cause cell damage and delivery failure. Chitosan (CS) is a potential cationic bio-derived polymer whose chemical structures can be modified to fine-tune the charges as well as the add-on functions. The present work demonstrates (i) the decoration of a nucleic acid sequence-like brush structure on CS to allow the specific interaction with DNA and (ii) delivery into the cell. By simply applying mercaptoacetic acid as the chain transfer agent, the grafting of poly(hydroxyethyl methacrylate) (PHEMA) containing Thy (P(HEMA-Thy)) on CS is possible. The brush-like P(HEMA-Thy) leads Thy moieties to be in sequences. The Thy sequences perform as poly[T] for the specific interaction with ssDNA. The synergistic effect of CS and Thy sequences, i.e., electrostatic and base pairing interactions, results in an effective and efficient binding with ssDNA as well as significant delivery, especially in cellular uptake and cell viability. The use of CS in combination with Thy sequences in brush-like structures on CS is a model for other polysaccharides to be conjugated with the as-designed nucleic acid sequences for potential gene delivery.

Dry Formulations Enhanced Mucoadhesive Properties and Reduced Cold Chain Handing of Influenza Vaccines.

To alleviate concerns in health security, emergency flu vaccine stockpiles are required for ensuring rapid availability of vaccines when needed. Cold chain preservation, at high cost and risk, is necessary to maintain vaccine efficacy. This study aimed to develop a dry, easily storable formula for influenza vaccine preparation. The formulation with mucoadhesive properties is expected to facilitate rapid delivery via nasal administration. Chitosan, a cationic polymer, was used as cryo-protectant and to promote mucoadhesion. Optimal concentrations and molecular weights of chitosan polymers were screened, with short chain chitosan (10 kDa) being most suitable. H1N1 dry powder, in different formulations, was prepared via freeze-drying. A series of cryo-protectants, trehalose (T), chitosan (C), fetal bovine serum (FBS; F), or a combination of these (TCF), were screened for their effects on prolonging vaccine shelf life. Physicochemical monitoring (particle size and zeta potential) of powders complexed with mucin revealed that the order of cryo-protectant mixing during preparation was of critical importance. Results indicated that the TCF formula retains its activity up to 1 year as indicated by TCID50 analysis. This approach was also successful at prolonging the shelf life of H3N2 vaccine, and has the potential for large-scale implementation, especially in developed countries where long-term storage of vaccines is problematic.

Controllable encapsulation of α-mangostin with quaternized ß-cyclodextrin grafted chitosan using high shear mixing.

In this study, the inclusion complex formation between α-mangostin and water-soluble quaternized ß-CD grafted-chitosan (QCD-g-CS) was investigated. Inclusion complex formation with encapsulation efficiency (%EE) of 5, 15 and 75% can be varied using high speed homogenizer. Tuning %EE plays a role on physicochemical and biological properties of α-mangostin/QCD-g-CS complex. Molecular dynamics simulations indicate that α-mangostin is included within the hydrophobic ß-CD cavity and being absorbed on the QCD-g-CS surface, with these results being confirmed by Fourier transform infrared (FTIR) spectroscopy. Probing the release characteristics of the inclusion complex at various %EE (5%, 15% and 75%) in simulated saliva (pH 6.8) demonstrated that α-mangostin release rates were dependent on % EE (order 5% > 15% > 75%). Additionally, higher antimicrobial and anti-inflammation activities were observed for the inclusion complex than those of free α-mangostin due to enhance the solubility of α-mangostin through the inclusion complex with QCD-g-CS.

Encapsulation of lemongrass oil with cyclodextrins by spray drying and its controlled release characteristics.

Inclusion of the two isomers of citral (E-citral and Z-citral), components of lemongrass oil, was investigated within the confines of various cyclodextrin (α-CD, ß-CD and γ-CD) host molecules. Aqueous complex formation constants for E-citral with α-CD, ß-CD and γ-CD were determined to be 123, 185, and 204 L/mol, respectively, whereas Z-citral exhibited stronger affinities (157, 206, and 253 L/mol, respectively). The binding trend γ-CD > ß-CD > α-CD is a reflection of the more favorable geometrical accommodation of the citral isomers with increasing cavity size. Encapsulation of lemongrass oil within CDs was undertaken through shaking citral:CD (1:1, 1.5:1, and 2:1 molar ratio) mixtures followed by spray drying. Maximum citral retention occurred at a 1:1 molar ratio with ß-CD and α-CD demonstrating the highest levels of total E-citral and Z-citral retention, respectively. Furthermore, the ß-CD complex demonstrated the slowest release rate of all inclusion complex powders.

Phospholipid-chitosan hybrid nanoliposomes promoting cell entry for drug delivery against cervical cancer.

This study emphasizes the development of a novel surface modified liposome as an anticancer drug nanocarrier. Quaternized N,O-oleoyl chitosan (QCS) was synthesized and incorporated into liposome vesicles, generating QCS-liposomes (Lip-QCS). The Lip-QCS liposomes were spherical in shape (average size diameter 171.5±0.8nm), with a narrow size distribution (PDI 0.1±0.0) and zeta potential of 11.7±0.7mV. In vitro mucoadhesive tests indicated that Lip-QCS possesses a mucoadhesive property. Moreover, the presence of QCS was able to induce the cationic charge on the surface of liposome. Cellular internalization of Lip-QCS was monitored over time, with the results revealing that the cell entry level of Lip-QCS was elevated at 24h. Following this, Lip-QCS were then employed to load cisplatin, a common platinum-containing anti-cancer drug, with a loading efficiency of 27.45±0.78% being obtained. The therapeutic potency of the loaded Lip-QCS was investigated using a 3D spheroid cervical cancer model (SiHa) which highlighted their cytotoxicity and apoptosis effect, and suitability as a controllable system for sustained drug release. This approach has the potential to assist in development of an effective drug delivery system against cervical cancer.

A comparison of eugenol and menthol on encapsulation characteristics with water-soluble quaternized ß-cyclodextrin grafted chitosan.

Two guest molecules (eugenol and (-)-menthol) were investigated on inclusion complex formation with water-soluble quaternized ß-CD grafted with chitosan (QCD-g-CS). The inclusion complexes were prepared at varying mole ratios between eugenol or (-)-menthol and ß-CD (substituted on QCD-g-CS) by a conventional shaking method and obtained as solid powder by freeze-drying process. The results showed that encapsulation efficiency %EE decreased with increasing of initial eugenol or (-)-menthol loading whereas %loading increased with increasing of initial eugenol or (-)-menthol loading. The results indicated that inclusion complex formation between eugenol and QCD-g-CS was more favorable than that of (-)-menthol. To clarify this mechanism, molecular dynamics simulations were performed to explore their binding energy, solvation energy and total free energy of those complexes. It was found that the total free energy (ΔG) of eugenol and (-)-menthol against QCD-g-CS (mole ratio of 1) in water-explicit system were -2108.91 kJ/mol and -344.45 kJ/mol, respectively. Moreover, molecular dynamic simulation of eugenol absorbed on surface QCD-g-CS (-205.73 kJ/mol) was shown to have a higher negative value than that of (-)-menthol on QCD-gCS (3182.31 kJ/mol). Furthermore, the release characteristics of the encapsulated powder were also investigated in simulated saliva pH 6.8 at 32 °C. The results suggested that (-)-menthol had higher release rate from the complexes than eugenol. In all cases, the release characteristics for those guest molecules could be characterized by the limited-diffusion kinetics.

Co-solvation effect on the binding mode of the α-mangostin/ß-cyclodextrin inclusion complex.

Cyclodextrins (CDs) have been extensively utilized as host molecules to enhance the solubility, stability and bioavailability of hydrophobic drug molecules through the formation of inclusion complexes. It was previously reported that the use of co-solvents in such studies may result in ternary (host:guest:co-solvent) complex formation. The objective of this work was to investigate the effect of ethanol as a co-solvent on the inclusion complex formation between α-mangostin (α-MGS) and ß-CD, using both experimental and theoretical studies. Experimental phase-solubility studies were carried out in order to assess complex formation, with the mechanism of association being probed using a mathematical model. It was found that α-MGS was poorly soluble at low ethanol concentrations (0-10% v/v), but higher concentrations (10-40% v/v) resulted in better α-MGS solubility at all ß-CD concentrations studied (0-10 mM). From the equilibrium constant calculation, the inclusion complex is still a binary complex (1:1), even in the presence of ethanol. The results from our theoretical study confirm that the binding mode is binary complex and the presence of ethanol as co-solvent enhances the solubility of α-MGS with some effects on the binding affinity with ß-CD, depending on the concentration employed.

The effect of surfactant composition on the chemical and structural properties of nanostructured lipid carriers.

Fine-tuning the nanoscale structure and morphology of nanostructured lipid carriers (NLCs) is central to improving drug loading and stability of the particles. The role of surfactant charge on controlling the structure, the physicochemical properties and the stability of NLCs has been investigated using three surfactant types (cationic, anionic, non-ionic), and mixed surfactants. Either one, a mixture of two, or a mixture of three surfactants were used to coat the NLCs, with these classified as one, two and three surfactant systems, respectively. The mixed (two and three) surfactant systems produced smaller NLC particles and yielded NLCs with lower crystallinity than the one surfactant system. The combined effects of the ionic and the non-ionic surfactants may play a key role in assisting the lipid-oil mixing, as well as maintaining colloidal repulsion between NLC particles. In contrast, for the three surfactant system, the lipid-oil mixture in the NLCs appeared less homogenous. This was also reflected in the results of the stability study, which indicated that NLC particle sizes in two surfactant systems appeared to be retained over longer periods than for other surfactant systems.

Influence of curcumin-loaded cationic liposome on anticancer activity for cervical cancer therapy.

The delivery of curcumin has been explored in the form of liposomal nanoparticles to treat various cancer cells. Since curcumin is water insoluble and an effective delivery route is through encapsulation in liposomes, which were modified with three components of DDAB, cholesterol and non-ionic surfactant. The purpose of this study was to establish a critical role of DDAB in liposomes containing curcumin at cellular response against two types of cell lines (HeLa and SiHa). Here, we demonstrate that DDAB is a potent inducer of cell uptake and cell death in both cell lines. The enhanced cell uptake was found on DDAB-containing liposome, but not on DDAB-free liposome. However, the cytotoxicity of DDAB-containing liposomes was high and needs to be optimized. The cytotoxicity of liposomal curcumin was more pronounced than free curcumin in both cells, suggesting the benefits of using nanocarrier. In addition, the anticancer efficiency and apoptosis effect of the liposomal curcumin formulations with DDAB was higher than those of DDAB-free liposomes. Therefore curcumin loaded liposomes indicate significant potential as delivery vehicles for the treatment of cervical cancers.